For centuries, light microscopy has been the main tool for studying cells under physiological conditions. In the 19th century, Ernst Abbe formulated the resolution limitation of the light microscope as approximately half the light wavelength, or 200-300 nanometers. Abbe's theory is discussed in detail in Born & Wolf, Principles of Optics (7th ed.) (1999), incorporated herein by reference, at pp. 467 ff. Electron microscopy (EM), on the other hand, can reveal nanometer scale details of the cellular structure because the wavelength associated with an accelerated electron is correspondingly smaller than that of a visible photon. However, EM has inherent limitations due to the heavy sample preparation involved, which prohibits investigating cells non-invasively. Many outstanding questions in cell biology could be answered if light microscopy provided the nanometer level resolution afforded by electron microscopy.
In recent years, several approaches have been developed to surmount the diffraction barrier in fluorescence microscopy, thereby representing a paradigm shift from the resolution limit formulated by Abbe. However, the current applications of these techniques are, in many cases, limited by specific technological constraints. First, in all approaches, the fluorescent light detected is very weak, which demands a correspondingly long exposure time, itself limited by photobleaching. Second, saturation techniques, such as stimulated emission depletion and structured illumination require a high level of excitation power to be delivered to the sample, which raises the issue of photo-damage and ultimately limits the safe exposure time. Third, approaches based on stochastic photo-switchable dyes operate based on the prior assumption of a sparse distribution of fluorescent molecules, which limits applications to dynamic imaging and long-time investigation.
The foregoing limitations may be overcome if the nanoscale cell structure and dynamics information is accessed via intrinsic contrast, i.e., without exogenous agents, such as fluorescent dyes. The great obstacle in this case becomes the fact that, generally, cells of single- and multi-cellular organisms do not absorb or scatter light significantly, i.e., they are essentially transparent, or phase objects. The phase contrast (PC) method of Zernike, Science 121, p. 345 (1955), which is incorporated herein by reference, represented a major advance in intrinsic contrast imaging, as it revealed inner details of transparent structures without staining or tagging. In PC, a phase shift of π/2 is introduced between the scattered and unscattered light, which makes the two interfere with greater contrast at the image plane. While PC is sensitive to minute optical path changes in the cell, down to the nanoscale, the information retrieved is qualitative, i.e., it does not provide the actual phase delay through the sample.
The intensity of light scattered by a particle as a function of the angle between the incident illumination and the scattered wave, and, more particularly, as a function of the incident wavelength and polarization, depends on the dimensions, morphology, optical susceptibility (or refractive index) and orientation of the scattering particle.
Elastic (static) light scattering (ELS) has made a broad impact in understanding inhomogeneous matter, from atmosphere and colloidal suspensions to rough surfaces and biological tissues. In ELS, by measuring the angular distribution of the scattered field, one can infer noninvasively quantitative information about the sample structure (i.e. its spatial distribution of refractive index). Dynamic (quasi-elastic) light scattering (DLS) is the extension of ELS to dynamic inhomogeneous systems. The temporal fluctuations of the optical field scattered at a particular angle by an ensemble of particles under Brownian motion relates to the diffusion coefficient of the particles. Diffusing wave spectroscopy integrates the principle of DLS in highly scattering media. More recently, dynamic scattering from a probe particle has been used to study the mechanical properties of the surrounding complex fluid of interest. Thus, microrheology, in which viscoelastic information is retrieved over various temporal and length scales, remains a subject of intense current research especially in the context of cell mechanics.
Light scattering techniques have the benefit of providing information that is intrinsically averaged over the measurement volume. However, it is often the case that the spatial resolution achieved is insufficient. “Particle tracking” microrheology alleviates this problem by measuring the particle displacements in the imaging (rather than scattering) plane. However, the drawback in the case of particle tracking is that relatively large particles are needed such that they can be tracked individually, which also limits the throughput required for significant statistical average.
The use of angular light scattering (ALS) or light scattering spectroscopy (LSS), generally, as techniques for studying the features of individual particles, and of particles in the aggregate, has a long history. Recent application to intact cells is the subject of Wilson et al., Mie theory interpretations of light scattering from intact cells, Opt. Lett., vol. 30, pp. 2442-44 (2005), while coherent techniques, using reference beams of varying degrees of coherence, have been applied to ALS, as described, for example, in Hillman, et al., Microscopic particle discrimination using spatially-resolved Fourier-holographic light scattering angular spectroscopy, Opt. Express, vol. 14, pp. 11088-11102 (2006), both of which references are incorporated herein by reference. All prior art coherent light scattering measurements have entailed measurements in the Fourier plane, such that each angle must be detected separately, by a distinct detector element or set of detector elements.